title: Vitamin D status assessed by a validated HPLC method: within and between variation in subjects supplemented with vitamin D3.
abstract: OBJECTIVE
  The aim of this study was to develop and validate a high-pressure liquid chromatography (HPLC) method for assessing vitamin D status as 25-hydroxyvitamin D(2) (S-25OHD(2)) and 25-hydroxyvitamin D(3) (S-25OHD(3)) in serum.
  
  
  METHODS
  We assessed the within- and between-subject variation of vitamin D status in serum samples from four different dietary intervention studies in which subjects (n = 92) were supplemented with different doses of vitamin D(3) (5-12 microg/day) and for different durations (4-20 months).
  
  
  RESULTS
  The HPLC method was applicable for 4.0-200 nmol S-25OHD/L, while the within-day and between-days variations were 3.8 % and 5.7 %, respectively. There was a concentration-dependent difference between results obtained by a commercial radioimmunoassay and results from the HPLC method of -5 to 20 nmol 25OHD/L in the range 10-100 nmol 25OHD/L. The between-subject variation estimated in each of the four human intervention studies did not differ significantly (p = 0.55). Hence, the pooled standard deviation was 15.3 nmol 25OHD(3)/L. In the studies with 6-8 samplings during 7-20 months of supplementation, the within-subject variation was 3.9-7.2 nmol 25OHD(3)/L, while vitamin D status was in the range 47-120 nmol/L.
  
  
  CONCLUSIONS
  The validated HPLC method was applied in samples from human intervention studies in which subjects were supplemented with vitamin D(3). The estimated standard deviation between and within subjects is useful in the forthcoming decision on setting limits for optimal vitamin D status.
authors.affiliation: National Food Institute, Technical University of Denmark, Søborg, Denmark. jeja@food.dtu.dk
authors.forename: Jette
authors.initials: J
authors.lastname: Jakobsen
authors.forename: Anette
authors.initials: A
authors.lastname: Bysted
authors.forename: Rikke
authors.initials: R
authors.lastname: Andersen
authors.forename: Teressa
authors.initials: T
authors.lastname: Bennett
authors.forename: Christine
authors.initials: C
authors.lastname: Brot
authors.forename: Susanne
authors.initials: S
authors.lastname: Bügel
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Cashman
authors.forename: Eberhard
authors.initials: E
authors.lastname: Denk
authors.forename: Mary
authors.initials: M
authors.lastname: Harrington
authors.forename: Birgit
authors.initials: B
authors.lastname: Teucher
authors.forename: Thomas
authors.initials: T
authors.lastname: Walczyk
authors.forename: Lars
authors.initials: L
authors.lastname: Ovesen
mesh_terms: D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D002112:Calcifediol / Q000008:administration & dosage* / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol